Studying the dynamic movement and interactions of proteins inside living cells is critical for a better understanding of cellular mechanisms and functions. Traditionally this has been done by in vitro labelling of proteins with fluorescent and other molecular probes, followed by transfer of the labelled protein into a live cell for real time monitoring, using advanced imaging techniques including confocal microscopy (G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif., 1996).
Recent advances in genetic engineering have made it possible to directly generate fluorescently labelled proteins in living cells, or even live animals, by fusion of fluorescent proteins such as GFP (Green Fluorescent Protein) to the protein of interest (R. Y. Tsien, Annu. Rev. Biochem. (1998) 57: 509). Although this technique may be a quite powerful method of visualizing proteins in vivo, the fusion of GFP or other fluorescent proteins with a target protein may affect the target protein's biological and cellular activities, due to the relatively large size of the fusion (i.e. 27 KDa for GFP). Furthermore, there are currently few fluorescent proteins available, thereby limiting the number of colours that can be used to “tag” a protein in vivo. As well, this strategy is limited to labelling a target protein only with protein molecular labels, typically fluorophores, but not other molecular probes such as small molecule probes.
In order to address some of these problems, Tsien and colleagues recently described a novel method which allows efficient labelling of proteins in vivo using cell-permeable organoarsenic compounds (B. A. Griffin, S. R. Adams and R. Y. Tsien, Science (1998) 281: 269). This approach requires insertion of an alpha-helical -CCXXCC-motif into the target protein, typically at the N- or C-terminus or at a surface-exposed region of the protein, for covalent binding with the organoarsenic compound. The introduction of such a motif has the potential to disrupt the native folding of the target protein. Furthermore, this method may result in background labelling, making it difficult to detect labelled target protein if expressed at low levels.
Johnsson and colleagues described enzyme-catalysed, in vivo labelling of proteins fused to the human DNA repair protein hAGT (A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel and K. Johnsson, Nat. Biotechnol. (2003) 21:86-89). This approach also results in the introduction of a macromolecular fusion into the target protein, potentially perturbing the native function of the protein.
Other in vivo-compatible chemical reactions, including the ketone-hydrazine reaction (Mahal, L. K., Yarema, K. J. & Bertozzi, C. R. Science (1997) 276:1125-1128; Zhang, Z. W., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz, P. G. Biochemistry (2003) 42:6735-6746), and the Staudinger reaction (Saxon, E. & Bertozzi, C. R. Science (2000) 287:2007-2010), have been successfully used for in vivo labelling of biomolecules. However, these methods require the introduction of non-natural occurring functionalities into the target biomolecule in vivo, and thus are useful for only limited applications, for example cell surface engineering and labelling of membrane proteins.
Methods for protein labelling based on the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae or Escherichia coli have been described (Chin, J. W., Cropp, T. A., Anderson, J. C., Mukherji, M., Zhang, Z. & Schultz, P. G. Science (2003) 301:964-967; Kiick, K. L., Saxon, E., Tirrell, D. A. & Bertozzi, C. R. Proc. Natl. Acad. Sci. USA (2002) 99:19-24). These methods may be complicated to use in that they require genetic manipulation of the host cellular expression system used to express the target protein.